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Series GSE58259 Query DataSets for GSE58259
Status Public on Dec 22, 2014
Title RNA-binding protein AUF1 promotes myogenesis by regulating MEF2C expression levels
Organism Mus musculus
Experiment type Other
Summary The mammalian RNA-binding protein AUF1 (AU-binding factor 1, also known as heterogeneous nuclear ribonucleoprotein D, hnRNP D) binds to numerous mRNAs and influences their post-transcriptional fate. Given that many AUF1 target mRNAs encode muscle-specific factors, we investigated the function of AUF1 in skeletal muscle differentiation. In mouse C2C12 myocytes, where AUF1 levels rise at the onset of myogenesis and remain elevated throughout myocyte differentiation into myotubes, RIP (RNP immunoprecipitation) analysis indicated that AUF1 binds prominently to Mef2c (myocyte enhancer factor 2c) mRNA, which encodes the key myogenic transcription factor Mef2c. By performing mRNA half-life measurements and polysome distribution analysis, we found that AUF1 associated with the 3’UTR of Mef2c mRNA and promoted Mef2c translation without affecting Mef2c mRNA stability. In addition, AUF1 promoted Mef2c gene transcription via a lesser-known role of AUF1 in transcriptional regulation. Importantly, lowering AUF1 delayed myogenesis, while ectopically restoring Mef2c expression levels partially rescued the impairment of myogenesis seen after reducing AUF1 levels. We propose that Mef2c is a key effector of the myogenesis program promoted by AUF1.
Keywords: ribonucleoprotein complex; post-transcriptional gene regulation; muscle cell differentiation; myocytes; mRNA translation; mRNA stability; post-transcriptional gene regulation; transcriptome
Overall design C2C12 mouse myoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% serum (Invitrogen) and antibiotics (Invitrogen). Differentiation was induced on sub-confluent cultures by replacing the growth media (GM, DMEM with 10% FBS) with differentiation media (DM, DMEM with 2% horse serum). At various time points after differentiation, cells were harvested and RNA was extracted with phenol-chloroform and either saved as input samples for microarrys or subjected to AUF1 or IgG ribonucleoprotein immunoprecipitation. C2C12 cells cultured in GM and DM were lysed in 20 mM Tris-HCl at pH 7.5, 100 mM KCl, 5 mM MgCl2, and 0.5% NP-40 for 10 min on ice and centrifuged at 15,000 × g for 10 min at 4°C. The supernatants were incubated with protein-A Dynabeads beads coated with anti-AUF1 (Millipore) or with control IgG (Santa Cruz Biotechnology) antibodies for 2 hr at 4°C. The beads were washed with NT2 buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40), followed by incubation with 20 units of RNase-free DNase I for 15 min at 37°C to remove the DNA. The samples were then incubated for 15 min at 55°C with 0.1% SDS/0.5 mg/ml Proteinase K to digest proteins. The RNA from the IP samples was extracted using phenol-chloroform, precipitated, and used along with the RNA from the input samples for cDNA microarrays.
Contributor(s) Panda AC, Abdelmohsen K, Yoon J, Martindale JL, Yang X, Curtis J, Mercken EM, Chenette DM, Zhang Y, Schneider RJ, Becker KG, de Cabo R, Gorospe M
Citation(s) 24891619
Submission date Jun 05, 2014
Last update date Jun 22, 2020
Contact name Supriyo De
Organization name NIA-IRP, NIH
Department Laboratory of Genetics and Genomics
Lab Computational Biology & Genomics Core
Street address 251 Bayview Blvd
City Baltimore
State/province Maryland
ZIP/Postal code 21224
Country USA
Platforms (1)
GPL6885 Illumina MouseRef-8 v2.0 expression beadchip
Samples (44)
GSM1405270 AUF1 RNA Pulldown Day0 Replicate1
GSM1405271 AUF1 RNA Pulldown Day0 Replicate2
GSM1405272 AUF1 RNA Pulldown Day0 Replicate3
BioProject PRJNA251754

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Supplementary file Size Download File type/resource
GSE58259_Non-normalized_data.txt.gz 4.2 Mb (ftp)(http) TXT
GSE58259_RAW.tar 3.1 Mb (http)(custom) TAR
Raw data are available on Series record
Processed data included within Sample table

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